Rituximab plus chemotherapy is standard therapy for patients with non-Hodgkin B cell lymphoma, but often complete response or cure is not achieved. Toll-like receptor 9 agonist CpG oligodeoxynucleotides (CpG) can improve antibody-dependent cellular cytotoxicity and adaptive antitumor immune responses. Using a syngeneic murine B cell lymphoma expressing human CD20 (38C13-huCD20), we previously demonstrated that rituximab plus intratumoral CpG, but not systemic CpG, could eradicate up to half of 7-day established 38C13-huCD20 tumors. However, larger 10-day established tumors could not be cured with this regimen. We thus hypothesized that cytoreduction with cyclophosphamide (Cy) before immunotherapy might permit eradication of these more advanced tumor burdens. Pretreatment with Cy resulted in tumor eradication from 83% of animals treated with rituximab/CpG, whereas Cy/CpG or Cy/rituximab treatments only cured 30% or 17%, respectively (P<0.005). Tumor eradication depended on natural killer cells, but not T cells, macrophages, or complement. Only mice treated with Cy/rituximab/CpG partially resisted rechallenge with tumor cells. Foxp3 Treg and CD11bGr1 myeloid suppressor cells persisted within lymphoid organs after therapy, possibly influencing the ability to establish adaptive tumor immunity. In conclusion, cytoreduction with Cy permitted the cure of large, established lymphomas not otherwise responsive to rituximab plus intratumoral CpG immunotherapy.